Generation of a mouse model for Zfyve27, the gene which is mutated in hereditary spastic paraplegia (SPG33)

ZFYVE27 is a novel protein, which belongs to the FYVE finger family of proteins. Majority of the FYVE finger proteins serve as regulators of endocytic membrane trafficking by recruiting cytosolic proteins to the endosomal membrane. In a recent study, we have demonstrated that ZFYVE27 specifically bind to spastin and plays important role in vesicular transport process. More importantly, we identified a missense mutation p. G191V in ZFYVE27 in a German family with pure form of AD-HSP (SPG33). The mutated ZFYVE27 protein shows aberrant intracellular pattern in tubular structure and its interaction with spastin is severely affected. Our analysis suggests that the identified mutation in SPG33 family will most likely resultin a loss of function of the protein due to the misfolding of C-terminal FYVE finger domain therefore a knockout mouse will be an appropriate model system to study this disease. To generate loss of function knockout mouse we will use gene-targeting technology to disturb the endogenous Zfyve27 gene of the mouse through homologous recombination in embryonic stem (ES) cells. In the targeting vector, we will replace the endogenous gene with green fluorescent protein, which will enable us to determine the endogenous expression pattern of Zfyve27. This mouse model will serve as critical means to elucidate the molecular mechanism involved in SPG33 pathology and will also allow us to examine why the nerves degenerate in HSP. In future this mouse model can be used for testing and development of new therapeutic approaches for treatments of HSP disease

Abschlussbericht

Institute of Human Genetics, University of Goettingen, Goettingen, Germany

ZFYVE27 (Protrudin) is a novel protein, which belongs to the FYVE finger family of proteins. Majority of the FYVE finger proteins serve as regulators of endocytic membrane trafficking by recruiting cytosolic proteins to the endosomal membrane. In a recent study, we have demonstrated that mutation in ZFYVE27 causes hereditary spastic paraplegia subtype 33 (SPG33)1. ZFYVE27 has been shown to interact with Rab11 and to promote the intracellular membrane trafficking events by virtue of its FYVE (Fab1P, YOTB, Vac1P and EEA1) domain2.

Expression pattern of ZFYVE27 in the mouse central nervous system

As ZFYVE27 is implicated in HSP, an upper motor neuron disease therefore it is vital to study expression of ZFYVE27 in the central nervous system (CNS) including different divisions of the brain as well as in the spinal cord. To achieve this goal, we have generated a high quality polyclonal antibody against mouse ZFYVE27 (214-344 amino acids) fused with GST protein using rabbit as a host. The antiserum from each rabbit was affinity purified against the GST-ZFYVE27 fusion protein using HiTrap column (Amersham). The purified antibody was used for the Western blot analysis. A comprehensive expression analysis of ZFYVE27 in the total protein extract isolated from various tissues of mouse revealed a high level of expression primarily in the HSP affected tissues such as the brain, cerebellum and spinal cord. Although, a weak expression was also detectable in the testis and cross-reactivity was observed in the liver, which could be either an isoform variant of ZFYVE27 or a paralogous protein. In a similar study, Shirane and Nakayama also reported high expression of ZFYVE27 (Protrudin) in the CNS and a weaker expression in some other tissues2. Immunohistochemical studies tissue sections from various subdivisions of the brain revealed that ZFYVE27 localize in the neuronal cell bodies as well as in the axons of hippocampus, cerebellum and corpus callosum. Minimal expression was detectable in oligodendroglial cell bodies as well as in white matter. In the spinal cord, expression of ZFYVE27 was primarily in the cell bodies of motor neurons and to lesser extent in the somatosensory neurons. The predominant expression of ZFYVE27 in the motor neurons highlights its role in neurodegenerative disease.

Generation of loss-of-function mouse model for Zfyve27

To produce the loss of function mouse model for ZFYVE27, we employed a novel Bacterial Artificial Chromosome (BAC)mediated homologous recombination method to generate the knockout construct in C57BL/6 background. The BAC mediated homologous recombination technique is based upon the Red/GET recombination, in which the bacteriophage recombination genes are genetically engineered in E. coli to efficiently manipulate the BAC containing gene of interest with ease in short period of time. Firstly, we identified two BAC genomic clones (RPCIB731C16470Q2 and RPCIB731A01447Q2) from the mouse genomic DNA library RPCI-23, which is derived from the C57BL/6 mouse strain containing full genomic sequence of Zfyve27 and 50 kb flanking sequence from either end of the gene. The BAC recombination method involves only two step procedure (1. integration and 2. retrieval) to generate a knockout construct. For integration step, a PCR cassette consisting of neomycin/kanamycin (Neo/Kanr) resistance genes is amplified by a primer pair consisting of a 60 bp homologous sequence (at either end) from the genomic regions of Zfyve27, which we wanted to delete. We decided to delete exon 6 till exon 11 of Zfyve27, which contains all the important functional domains and motifs including the FYVE domain of ZFYVE27. As there were several ATG initiation regions predicted in Zfyve27 gene therefore deletion of either of the ATG region was risky and also not desirable. Moreover, the possibility of truncated transcript is also rare as such transcripts will be degraded by nonsense mediated decay system (personal experience). In next step, we retrieved a 5’ homologous arm of 6.1 kb and 3’ homologous arm of 5.6 kb into knockout construct containing thymidine kinase for negative selection. The generated knockout construct was linearized by Not I digestion and electroporated into ESC (cell line: TS3/C57BL/6). After selection (neomycin as positive selection marker and thymidine kinase as negative marker) for 10 days, the genomic DNA was isolated from 120 recombinant ESC clones and genomic Southern blot was performed with both 5’ and 3’ external probes. Screening of 60 ES clones led to identification of 9 recombinant clones. We have injected ES cells from two different clones into the blastocyst derived from the Balb/c and C57BL/6 albino strains. Currently, we have generated chimeric mice and we will breed the high percentage of chimeric mice for establishing the knockout mouse lines.

Conceivably the phenotype of these generated mouse models might mimic the pathological features of HSP, therefore will provide us with a valuable model system to elucidate the underlying cause for HSP etiology.